Proteinase Allostery and the Regulation of Blood Coagulation

蛋白酶变构和凝血调节

• 批准号: 8463606
• 负责人: Sriram Krishnaswamy
• 金额: $ 39.87万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8463606
• 关键词: Accounting; Active Sites; Address; Affect; Anticoagulants; Anticoagulation; Binding; Blood; Blood Clot; Blood coagulation; Calorimetry; Coagulation Process; Coenzymes; Color; Complement; Crystallography; Development; Disease; Distant; Enzyme Precursors; Enzymes; Equilibrium; Eye; Face; Fibrin; Fibrinogen; Fibrinolysis; Goals; Heart; Heating; Hemostatic function; Kinetics; Knowledge; Life; Ligand Binding; Ligands; Ligation; Light; Measurement; Methodology; N-terminal; Pathway interactions; Peptide Hydrolases; Physiological; Platelet Activation; Play; Process; Production; Property; Proteins; RNA; Reaction; Regulation; Research; Resolution; Role; Scheme; Series; Serine Protease; Site; Specificity; Structure; Testing; Therapeutic; Thermodynamics; Thrombin; Thrombomodulin; Thrombus; Titrations; Translating; Ursidae Family; Variant; Vascular Diseases; aptamer; base; human disease; insight; meizothrombin; novel; novel strategies; phrases; prevent; response; structural biology; therapeutic target

DESCRIPTION (provided by applicant): Thrombin, the terminal serine proteinase of the blood coagulation cascade, plays a pivotal role in thrombus formation as well as its regulation. These multiple and sometimes opposing roles of thrombin in coagulation are influenced by two exosites (ABE1 and ABE2) on opposite faces of the enzyme and cofactors or ligands that bind to these or other sites to allosterically regulate the proteinase. Major gaps and inconsistencies remain in the mechanistic understanding of how thrombin allostery is achieved and translates into regulated function. We broach the problem with the unexpected finding that thrombin can readily and reversibly interconvert along a continuum of zymogen-like and proteinase-like states depending on the complement of ligands bound to the enzyme. We propose that these interconversions lie at the heart of thrombin allostery. Our studies center on the finding that fragment 1.2 (F12), the authentic protein ligand for ABE2, thermodynamically favors zymogen- like forms. We will now employ titration calorimetry to establish the thermodynamic basis by which thrombomodulin (TM), which binds to ABE1, selectively stabilizes and favors proteinase-like forms to oppose the effects of F12. We also find that meizothrombin (mIIa), produced as an intermediate during thrombin formation, is particularly zymogen-like because of covalent linkage between F12 and the proteinase domain. We will employ a stopped-flow kinetic approach to examine the distribution of mIIa between zymogen-like and proteinase-like forms. Although the F2 region within F12 is expected to contact ABE2, we now propose a novel function for the Ca2+ stabilized structure of the putatively distant F1 in enforcing the ability of F12 to favor zymogen-like forms. Based on these mechanistic studies, we will study the regulation of thrombin function by opposing effects of ABE1 binding by different ligands and F12 binding to ABE2, with an eye to explaining the apparent differences in specificity ascribed to mIIa and various anticoagulant thrombins. We hypothesize that their altered specificity lies in their significantly zymogen-like character that can be variably rescued by the binding of ligands and substrates. These concepts also provide the appropriate framework for the development of novel aptamer probes that can modulate the distribution of thrombin between zymogen-like and proteinase- like states and thereby regulate its specificity with therapeutic potential. Finally, based on our recent successful entry into the structural biology arena, we propose the long term goal of solving a series of novel structures to establish the structural basis for the zymogenizing function of F12. Our strategies bring fresh and unifying concepts to the important problem of thrombin allostery. We anticipate our findings to shed new light on the mechanisms at play in regulating thrombin function in normal hemostasis and in disease states. Our findings have the potential to reveal new strategies for therapeutic targeting of this enzyme in thrombotic and vascular disease.

描述（由申请人提供）：凝血酶是血液凝结级联的末端丝氨酸蛋白酶，在血栓形成及其调节中起关键作用。凝血酶在凝结中的这些多重且有时相反的作用受两个外os岩（ABE1和ABE2）的影响，在酶的相反面部，辅助因子或辅助因子或配体与这些或其他位点结合以分配蛋白酶。对凝血酶变构的实现方式并转化为受调节功能的机械理解，主要差距和不一致仍然存在。我们提出了这个问题，即意外发现，凝血酶可以轻松而可逆地沿着酶机样和蛋白酶样状态的连续体相互互换，这取决于与酶结合的配体的补体。我们建议这些相互转换位于凝血酶变构的核心。我们的研究集中在发现片段1.2（F12）的发现，即Abe2的真实蛋白质配体，热力学上有利于Zymogen类型。现在，我们将采用滴定量热法来建立与ABE1结合的血小板结合蛋白（TM）的热力学基础，有选择地稳定并有利于蛋白酶样形式，以反对F12的作用。我们还发现，由于F12和蛋白酶结构域之间的共价连接，在凝血酶形成过程中作为中间体产生的梅氏蛋白酶（MIIA）尤其类似酶。我们将采用一种停止的流动动力学方法来检查MIIA在类似酶原和蛋白酶样形式之间的分布。尽管有望在F12中的F2区域接触ABE2，但我们现在提出了一个新的功能，用于在强制实施F12的能力偏向于Zymogen类似Zymogen类型的能力方面的Ca2+稳定结构。基于这些机械研究，我们将通过不同的配体与ABE1结合的影响和F12结合与ABE2结合，研究凝血酶功能的调节，并着眼于解释特异性在MIIA和各种抗凝血栓素中的明显差异。我们假设它们的特异性改变在于它们的明显类似酶原的特征，这可以通过配体和底物的结合而可变地挽救。这些概念还为开发新型适体探针的开发提供了适当的框架，这些探针可以调节酶原样和蛋白酶类似状态之间凝血酶的分布，从而以治疗潜力调节其特异性。最后，基于我们最近成功进入结构生物学领域，我们提出了解决一系列新型结构的长期目标，以建立F12的Zymogenation功能的结构基础。我们的策略将新鲜而统一的概念带入了凝血酶变构的重要问题。我们预计我们的发现将为调节正常止血和疾病状态的凝血酶功能的机制提供新的启示。我们的发现有可能揭示该酶在血小板和血管疾病中的治疗性靶向的新策略。